IL-23 is a heterodimeric molecule comprising a p19 subunit and a p40 subunit that are two disulfide-linked. IL-23 is speculated to play an essential role in chronic inflammation and autoimmune diseases in humans. Mice lacking p19 exhibit a decreased pro-inflammatory response to experimental autoimmune encephalomyelitis, inflammatory bowel disease and collagen-induced arthritis. While IL-23 per se cannot induce the differentiation of naïve CD4 T-cells into Th-17 cells in vitro, the differentiation of Th17 cells in vivo may require IL-23. The observed protective effect in p19-deficient mice may relate to the lack of differentiation of Th17 cells. This is consistent with recent report that IL-23 synergies with Th17 cell differentiation cytokines including IL-1, IL-6 and TGF-β to induce expression of IL-17.
IL-23 exerts its biological activities by binding to IL-23 receptor (IL-23R). IL-23R comprises an IL-23Rα subunit and an IL-12Rβ1 subunit. When IL-23 binds to IL-23R, it leads to intracellular signaling including phosphorylation of STAT1, STAT3, STAT4 and STAT5. IL-23R is expressed on T-cells, NK cells, monocytes, and dendritic cells and its expression pattern corresponds with the ability of these cells to respond to IL-23.
Human IL-23Rα mRNA is 2.8 kb long and contains 11 exons (NM_144701). The translated full-length IL-23Rα protein is a type-I transmembrane protein (629 amino acids) and contains at least three (3) known structural domains: (1) a signal peptide domain; (2) an extracellular region containing a N-terminal fibronectin III-like domain; and (3) a 253 amino acid residue cytoplasmic domain with three (3) potential tyrosine phosphorylation sites.
Christi Parham et al. first discovered the genomic and structural organization of the IL-23R (composed of an IL-23α subunit and an IL-12Rβ1 subunit). While IL-23 is shown to bind to IL-23R and mediates Jak-STAT cell signaling, Parham explicitly stated their inability to demonstrate human IL-23R-Ig and soluble human IL-23Rα-V5-His6 (composed of the entire extracellular domain—amino acids 1-353) as effective antagonists for human IL-23R. Daniel J. Cua et al. disclose treatment methods for multiple sclerosis, neuropathic pain, and inflammatory bowel disorders using antibodies against IL-23 and its receptor. Contrary to Parham's statement, Cua proposes using a soluble receptor based on the extracellular region of a subunit of the IL-23 receptor (PCT/US2004/003126) as an antagonist. A recombinant human IL-23Rα Fc chimeric protein is commercially available (R&D Systems) and claimed to have the ability to inhibit IL-23 induced IL-17 secretion in a mouse splenocytes system. It remains unclear as to whether any of these proposed soluble IL-23Rαs may in fact exist in vivo as a naturally-occurring protein, let alone the possibility that such soluble IL-23Rs may possess ability to block IL-23Rα mediated cell signaling. To this end, Daniel J. Cua et al. (PCT/US2004/003126) failed to provide any evidence that a soluble IL-23 receptor can indeed block IL-23 mediated cell signaling as well as inhibiting Th17 producing cells.
Recent evidence suggests that IL-23Rα gene may undergo extensive alternative splicing. There are at least twenty-four (24) potential gene transcripts for IL-23Rα. From these IL-23Rα alternatively spliced mRNA sequences, there appears at least four (4) deduced putative translated proteins: (1) a short premature IL-23Rα extracellular peptide; (2) a possible soluble form of IL-23Rα lacking a transmembrane/intracellular domain; (3) a full-length IL-23Rα with truncated extracellular region; and (4) a non-responsive membrane-bound receptor isoform of IL-23Rα with deletion in intracellular signaling components.
Although many gene transcripts for IL-23 Rα (i.e., IL-23R splice variants) are suggested, it is important to point out that their actual existence in vivo is presently unknown. There is little information regarding whether any of the deduced IL-23Rα translated products actually exist in vivo, let alone the function of these IL-23Rα protein variants, if any.
Accordingly, there is a continuing need for a diagnostic assay that detects a measurable level of IL-23Rα variants in a biological sample in a mammal, specifically an accurate ELISA that measures an isoform of IL-23Rα. The assay would enable the assessment of a pathological role of IL-23Rα using biological samples obtained from patients. The present inventors overcome the prior art deficiency and discovered an ELISA assay for quantifying a naturally-occurring soluble truncated form of IL-23Rα (i.e., Δ9 IL-23Rα) in plasma. The present ELISA reveals that a particular form (i.e., Δ9 IL-23Rα) of IL-23Rα constitutes a major spliced variant form of IL-23Rα in plasma and that its level correlates with inflammatory bowel diseases such as Crohn's disease.